Structural determinants for specific recognition by T4 endonuclease V

摘要: DNA glycosylases catalyze the scission of N-glycosyl bond linking either a damaged or mismatched base to sugar phosphate backbone. T4 endonuclease V is glycosylase/apurinic (AP) lyase that specific for UV light-induced cis-syn pyrimidine dimers. As proposed transition state analog/inhibitor glycosylases, phosphoramidite derivative containing pyrrolidine residue has been synthesized. The binding this duplex was analyzed by gel mobility shift assays and resulted in single stable complex reduced an apparent Kd 17 nM. To assess importance positive charge binding, studies using other non-cleavable substrate analogs were performed. Wild type shows 8-fold decreased affinity tetrahydrofuran as compared with residue, demonstrating significance recognition. A 2-fold increase AP site observed. Similar catalytically compromised mutants (E23Q E23D) demonstrate altered affinities well sites. This approach provided insight into structural mechanism which lesions are targeted protein determinants required recognition V.